1. Field of the Invention
The instant disclosure pertains to a method for synthesizing oligonucleotides. In particular, this disclosure pertains to a method for deprotecting oligonucleotides which results in a higher yield of crude oligonucleotide product with greatly improved purity.
2. Background
Oligonucleotides find enormous utility in a number of biological applications. For example, oligonucleotide sequences form duplexes with complementary oligonucleotide targets and can be used as molecular biological probes in genome research and in clinical diagnostic applications involving disease causing genes. In this application, materials containing a nucleic acid to be detected are brought in contact with an oligonucleotide probe which forms a duplex with its complementary nucleic acid sequence. The duplex is then detected using various analytical techniques.
Oligonucleotides also find utility as primers for various polymerase applications such as the polymerase chain reaction (“PCR”). In PCR, an oligonucleotide primer is added to a sample containing single stranded template nucleic acid fragments in the presence of an enzyme and mononucleotides. Starting at the primer, the enzyme builds a nucleic acid strand which is complementary to the template nucleic acid. The reaction is carried out several times in succession where in each cycle the newly built strand is amplified.
Oligonucleotides have also been used as a combinatorial discovery tool to design oligonucleotide sequences that act as competitive inhibitors of certain disease causing and unwanted proteins. In this application, also known as the “aptamer approach,” the high affinity of a particular protein of interest with a specific oligonucleotide sequence present in a random pool of millions of randomly synthesized oligonucleotide sequences is determined. This technique has been instrumental in developing novel oligonucleotide inhibitors of various proteins such as thrombin.
Oligonucleotides have even been found to have the capability to modulate gene expression at the messenger RNA level (“antisense”) or at the DNA level (“antigene” or “triplex”). The efficacy of a large number of oligonucleotide candidates is presently under clinical evaluation.
Given the significant therapeutic, diagnostic, and research utility of oligonucleotides, there is a need to prepare them in large quantities easily, quickly and at low cost. Phosphite triester and H-phosphonate chemistries are commonly used to prepare oligonucleotides on a solid support or substrate. Large scale commercial DNA synthesizers that employ phosphite triester chemistry, has made the production of multi-kilo grams of oligonucleotides possible.
Nucleosides used in large scale synthesis of oligonucleotides on a solid phase by phosphoramidite chemistry use are protected with suitable groups that prevent formation of side products during oligonucleotide synthesis. The reactive exocyclic amine groups found on the nucleobases in monomer building blocks are generally protected with benzoyl, isobutyrl, phenoxyacetyl, and acetyl protecting groups, while the phosphate groups are usually protected as 2-cyanoethyl phosphoramidites. Such protective groups are easily removed after completion of the oligonucleotide synthesis by treatment with a concentrated solution of is ammonium hydroxide.
Oligonucleotide synthesis begins by attaching a suitably protected, 5′-O-dimethoxytritylated nucleoside to a substrate. The 3′-hydroxyl group of the protected nucleoside is connected via a succinic ester linkage to the substrate. The most commonly used substrates are inorganic materials such as long chain glass or organic supports such as polystyrene; however, other supports such as polyamide, cellulose, silica gel, and polyethylene glycols, are also used in the solid phase synthesis of oligonucleotides.
The oligonucleotide is assembled by sequential addition of 5′-dimethoxytritylated-3′nucleoside phosphoramidites to the unmasked 5′-hydroxy group of the first nucleoside loaded on to the support. This addition is catalyzed by a mildly acidic catalyst such as tetrazole or dicyanoimidazole. The corresponding phosphite triester (“PIII”) internucleotide linkage is then converted to a more stable phosphate triester (“PV”) by oxidation with iodine or peroxides. “Capping” of any unreacted 5′-hydroxyl groups by converting them to corresponding esters is achieved by a brief exposure to capping reagents containing acetic anhydride. Next, removal of 5′-dimethoxytrityl group from the newly added nucleoside under mildly acidic conditions generates the 5′-hydroxyl group and completes the coupling cycle. Using this method, a coupling efficiency of greater than 99% in each coupling step can be achieved. Towards the end of oligonucleotide synthesis, the dimethoxytrityl group of the terminal nucleotide at the 5′-end is either left intact (“trityl-on”) or cleaved to give an oligonucleotide with free 5′-terminal hydroxyl group (“trityl-off”). The 5′-trityl group may be used as a lipophilic purification handle to purify the full length oligonucleotide bearing the trityl group from shorter and non-tritylated oligonucleotide species by reverse HPLC.
After completion of oligonucleotide synthesis, the succinic ester linkage is cleaved under alkaline conditions to release the oligonucleotide from the substrate in addition to the removal of protective groups from the nucleobases and the phosphate backbone. This is achieved by treating the substrate with a concentrated solution of ammonium hydroxide. This process usually takes about 24 hours at room temperature or about 6 hours at 55° C.
One of the major problems associated with stepwise coupling of mononucleotides phosphoramidites is the formation of shorter deletion sequences during oligonucleotide synthesis. This population of unwanted failure sequences (“n−1,” “n−2,” etc.) results from less than 100% coupling efficiency of the phosphoramidites, incomplete capping and oxidation or partial unmasking of the 5′-hydroxyl group before the initiation of the next coupling cycle.
Frequently observed, but least addressed are the so called “n+1” or “n+2” oligonucleotide impurities that are seen to elute immediately after the main oligonucleotide peak on HPLC columns. These unwanted impurities can comprise up to 6% of the total crude product cleaved from the substrate. Little is known about the nature and cause of these impurities. The elution of these impurities close to the main product makes the isolation of the purified oligonucleotide a difficult and time-consuming task and results in a poor recovery of the full length pure product.
As the above discussion suggests, improvements are still possible and desirable in the area of oligonucleotide synthesis. In particular, a method is needed for synthesizing oligonucleotides which results in a high yield of purer crude product. Ideally, such a method would not involve increased synthesis time. Preferably, such a method would also be economical to use. These and other concerns are addressed in greater detail below.